EP0149430A1 - Procédé pour la préparation d'ADN chimérique - Google Patents

Procédé pour la préparation d'ADN chimérique Download PDF

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Publication number
EP0149430A1
EP0149430A1 EP84810636A EP84810636A EP0149430A1 EP 0149430 A1 EP0149430 A1 EP 0149430A1 EP 84810636 A EP84810636 A EP 84810636A EP 84810636 A EP84810636 A EP 84810636A EP 0149430 A1 EP0149430 A1 EP 0149430A1
Authority
EP
European Patent Office
Prior art keywords
dna
virus
foreign gene
gene
plasmid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP84810636A
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German (de)
English (en)
Inventor
Normand Dr. University of Montreal Brisson
Linda Katleen Dr. Dixon
Thomas Dr. Hohn
Bruno Dr. Gronenborn
Jerzy Dr. Paszkowski
John Robert Dr. Penswick
Ingo Dr. Potrykus
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis AG
Original Assignee
Ciba Geigy AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ciba Geigy AG filed Critical Ciba Geigy AG
Publication of EP0149430A1 publication Critical patent/EP0149430A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation

Definitions

  • the present invention relates to a method for producing chimeric DNA, which consists of virus DNA from a plant virus and a foreign gene which is expressed in plant material.
  • plants with new or improved properties can be created.
  • essential aspects are, for example, an increase in the resistance of crop plants to diseases and pests, which can be brought about by induction or increased formation of protective substances or toxins in the plant, an increased resistance to crop protection agents such as insecticides, herbicides, fungicides and bactericides, and a beneficial change in the nutrient content of plants which can be induced by induction to produce valuable proteins or otherwise.
  • a particular aspect is the stimulation of plant material to produce non-plant, i.e. substances not formed in the original plants.
  • a corresponding influencing of plant material can take place, for example, by introducing new DNA, which can be chimeric DNA, it being possible for the plant material to be present in the form of cell cultures or propagation material of useful plants or as whole plants.
  • the present invention relates to a method for producing chimeric DNA, which consists in inserting a foreign gene that is expressed in plant material into the DNA of a plant virus.
  • the invention also relates to the use of the chimeric DNA for infecting plant material, a plant virus containing the chimeric DNA being used as the vector.
  • FIG. 1 Further aspects of the present invention relate to cauliflower mosaic virus which contains the chimeric DNA, and plant material which is infected with such a virus, the use of the chimeric DNA to infect plant material using such a virus as a vector and new ones Plasmids.
  • one expediently proceeds by removing one or more regions from the virus DNA, taking into account only those regions which are not required for the multiplication of the virus which harbors the chimeric DNA.
  • a foreign gene is then introduced into the remaining virus DNA.
  • the foreign gene is used in a suitable region of the virus DNA which does not impair the expression of the foreign gene and the reproductive capacity of the virus.
  • the foreign gene is advantageously introduced where a virus DNA region has been removed (deletion site), the inserted foreign gene replacing one or more of the removed virus DNA regions. If several deletion sites are present, several genes can also be inserted into the virus DNA, it proving to be particularly advantageous if one of these genes shows an easily detectable expression in the plants, which can be used as a label for the presence of the chimeric DNA.
  • the distance between the stop signal of the virus gene located immediately in front of the foreign gene used and the start signal of the foreign gene used is preferably kept as short as possible.
  • flanking sequences of the gene to be used are removed as completely as possible, which is expediently carried out enzymatically, for example with a suitable nuclease.
  • the reading frame of the virus DNA is maintained.
  • the foreign gene to be used must not have homologous ends and must be used in the correct sense of orientation.
  • the chimeric DNA which also forms an object of the present invention, can be used, using the plant viruses serving as virus DNA donors and containing the chimeric DNA, as vectors for infecting plant material, for example cell cultures or propagation material from useful plants or from Whole plants are used, the foreign gene used maintaining the existing ability for expression in the context of the present invention.
  • Representatives of the Cruciferae, Leguminosae, Rosaceae, Compositae and Solanaceae families are particularly suitable as useful plants.
  • Preferred virus DNA donors and vectors are DNA viruses or the DNA copy of a virus RNA, preferably caulimoviruses and especially original or recombinant strains of the cauliflower mosaic virus (CaMV, cauliflower mosaic virus).
  • CaMV cauliflower mosaic virus
  • the foreign gene to be inserted is expediently placed in the place of the removed ORF II.
  • the gene to be used can be derived from various organisms, for example bacteria.
  • a particularly well-suited gene is, for example, the DHFR gene (dihydrofolate reductase gene).
  • flanking sequences of the gene to be used are removed as completely as possible, preferably using a suitable enzyme, such as, for example, nuclease Bal 31.
  • a suitable enzyme such as, for example, nuclease Bal 31.
  • a further G follows the start signal ATG of the coding sequence, which is often the case above average, a NcoI restriction site (CCATGG) can be introduced with a coupler sequence that ends with CC at the 5 'end of the coding sequence, which acts as Clutch can be used.
  • This sequence is used to construct the chimeric CaMV vector.
  • the desired molecule can be selected using the Ncol sequence.
  • DHFR gene plasmid-encoded dihydrofolate reductase gene
  • the DHFR gene is inserted into the Cauliflower Mosaic Virus DNA instead of the distant ORF II region.
  • the foreign gene to be used which is encoded by the plasmid R67, represents the gene for the enzyme DHFR which is resistant to methotrexate and has 234 base pairs. Resistance to the folic acid antagonist methotrexate (4-amino-N -methylpteroyiglutamic acid) is an excellent indicator that makes the presence of expression in plant material easily recognizable.
  • plants of the Brassica rapa species are inoculated with cloned chimeric virus DNA.
  • the infection symptoms develop in the same period as in control plants which have been treated with the original virus DNA, which corresponds to the receiving site for the DHFR gene. It can be concluded from this that the presence of the DHFR gene does not adversely affect the replication of the virus.
  • virus particles are isolated from systemically infected leaves and the DNA obtained from this is broken down enzymatically.
  • the DNA fragment, which contains the DHFR gene is present in the expected molar proportion, from which it can be concluded that the foreign DNA is permanently preserved in the virus genome.
  • the stability of the gene used which is somewhat less pronounced in older parts of the plant than in younger parts of the plant, can be further improved by shortening the distance between the end of the viral gene upstream of the foreign gene and the beginning of the DHFR gene. It can thus be shown that even after two successive transfers of viruses from systemically infected plant tissues, the DHFR gene used is present and the DNA fragment which contains the DHFR gene is present in the expected molar proportion.
  • the expression of the foreign gene used can be detected with antibodies against the gene product and with functional tests and is expressed in the clearly detectable resistance of the plants infected with the chimeric DNA to methotrexate.
  • a Bam-HI fragment as used in Example 1 below, can be obtained by methods known per se, for example by treating a bacterial plasmid containing the DHFR gene, such as R67 or R388, with the restriction enzyme Bam-HI.
  • the plasmid pUCBX which is used in Example 1 below, can be prepared from the commercially available plasmid pUC8 by inserting an Xhol / EcoRI adapter into the EcoRI site.
  • the plasmid CaMV20-Bal 1 used in the following example 2 as the receiving site for the DHFR gene can be derived from the CaMV strain Win C M 4-184 by removing the region ORF II in a manner known per se by treatment with Bal 31 and introducing an XhoI coupling fragment into the interface.
  • Example 1 A Bam HI fragment (FIG. 2a) which contains the complete DHFR gene is isolated and fed into the plasmid pUC8X (FIG. 2b), into which an XhoI restriction site for the subsequent transfer of the DHFR gene has been introduced.
  • the plasmid .pJP74 is obtained in this way (FIG. 2c).
  • the isolated XhoI / SalI fragment of the plasmid pJP91 is inserted at the Xhol site of the plasmid pCaMV20-Bal 1 and the plasmid pCaMV-NBI (FIG. 3b) is thus obtained, which contains the DHFR gene inserted in the correct sense of orientation.
  • the expression (transcription and translation) of signal sequences of the original CaMV DNA is controlled.
  • Example 4 Plants of the species Brassica rapa are inoculated with viruses which contain one of the plasmids pCaMV-NB1 or pCaMV-NB2.
  • Leaf strips from plants infected in this way and from non-infected plants are in common solid and liquid Culture media containing methotrexate in various concentrations are cultivated. 4 days after moving the leaf strips into the culture media, there are clear differences on the leaf strips. From a concentration of methotrexate of 0.01 pM, only those leaf strips which have been infected with pCaMV-NBI or pCaMV-NB2 viruses show a normal appearance.
  • Example 5 Leaf strips, which have been cultivated in culture media containing methotrexate as described in Example 4 above, are transferred after a certain period of time to a corresponding culture medium without methotrexate.
  • the control tissues die after a residence time of 24 hours in a culture medium with concentrations of 0.01 to 0.2 pM methoxtrexate. After transfer to culture media without methotrexate, the tissues infected with pCaMV-NB1 or pCaMV-NB2 viruses form calli.
  • Example 6 Two plants of the Brassica rapa species are infected with pCaMV-NB1 or pCaMV20-Bal 1 viruses and sprayed with 15 ml of a solution of 50 ⁇ g methotrexate / ml. One week after treatment with methotrexate, the plants infected with pCaMV20-Bal 1 viruses died almost entirely, while the plants infected with pCaMV-NBl viruses showed no damage.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
EP84810636A 1984-01-04 1984-12-18 Procédé pour la préparation d'ADN chimérique Withdrawn EP0149430A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CH16/84 1984-01-04
CH1684 1984-01-04
CH3184 1984-01-05
CH31/84 1984-01-05

Publications (1)

Publication Number Publication Date
EP0149430A1 true EP0149430A1 (fr) 1985-07-24

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EP84810636A Withdrawn EP0149430A1 (fr) 1984-01-04 1984-12-18 Procédé pour la préparation d'ADN chimérique

Country Status (5)

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EP (1) EP0149430A1 (fr)
AU (1) AU3725485A (fr)
BR (1) BR8500010A (fr)
HU (1) HU193246B (fr)
IL (1) IL73993A0 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0242016A1 (fr) * 1986-01-23 1987-10-21 Agricultural Genetics Company Limited Modification de virus de plantes ou de leurs effets

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE74162T1 (de) * 1983-08-04 1992-04-15 Ciba Geigy Ag Verfahren zur erhaltung und vermehrung von defekten, nicht-infektioesen virusgenomen.

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0067553A2 (fr) * 1981-05-27 1982-12-22 National Research Council Of Canada Vecteur à base d'ARN de virus de plante ou une partie de celui-ci, procédé pour sa production, et une méthode de production d'un produit dérivé de gène, à l'aide de celui-ci

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0067553A2 (fr) * 1981-05-27 1982-12-22 National Research Council Of Canada Vecteur à base d'ARN de virus de plante ou une partie de celui-ci, procédé pour sa production, et une méthode de production d'un produit dérivé de gène, à l'aide de celui-ci

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol. 80, No. 15, August 1983 (Baltimore, USA) R.T. FRALEY et al "Expression of bacterial genes in plant cells" Seiten 4803-4807 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0242016A1 (fr) * 1986-01-23 1987-10-21 Agricultural Genetics Company Limited Modification de virus de plantes ou de leurs effets

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HU193246B (en) 1987-08-28
AU3725485A (en) 1985-07-18
BR8500010A (pt) 1985-08-13
IL73993A0 (en) 1985-04-30
HUT40161A (en) 1986-11-28

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Effective date: 19871121

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Inventor name: BRISSON, NORMAND, DR.UNIVERSITY OF MONTREAL

Inventor name: GRONENBORN, BRUNO, DR.

Inventor name: POTRYKUS, INGO, DR.

Inventor name: PENSWICK, JOHN ROBERT, DR.

Inventor name: PASZKOWSKI, JERZY, DR.

Inventor name: DIXON, LINDA KATLEEN, DR.

Inventor name: HOHN, THOMAS, DR.